This invention relates to a colorimetric method for the quantitative determination of gamma-glutamyl transpeptidase enzyme activity in a biological fluid, especially in human blood serum, and to compositions useful in the method.
Gamma-glutamyl transpeptidase is an enzyme which cleaves the C-terminal amino acid, glutamic acid of proteins and peptides. Serum gamma-glutamyl transpeptidase activity is elevated in patients with hepatobiliary tract disease. Extremely high values are usually associated with metastatic cancer of the liver and common bile duct obstruction due to neoplasms. High gamma-glutamyl transpeptidase values also have been reported in patients with various other liver diseases, neurologic disease and post-myocardial infarction. In hepatobiliary disease, the change in gamma-glutamyl transpeptidase activity is a more sensitive indicator than alkaline phosphatase and leucine aminopeptidase. The determination of gamma-glutamyl transpeptidase activity in serum has been shown to be a valuable aid in clinical diagnosis.
Gamma-glutamyl transpeptidase in biological materials is assayed by a transferase reaction, in which the enzyme acts catlytically upon a substrate of a synthetic peptide of L-glutamic acid, having a C-terminal glutamyl radical and a free carboxyl group, to transfer the glutamyl radical to a receptor having an L-amino acid moiety, including amino acids and peptides. The quantity of the cleavage product resulting from the transfer of the glutamyl radical from the synthetic peptide is determined as a measure of the activity of the enzyme.
The most commonly employed substrate for the assay of gamma-glutamyl transpeptidase is L-glutamyl-p-nitroanilide (J. Biol. Chem, 240, 338 (1965); Clin. Chem., 15, 124 (1969)). The cleavage product of the enzyme reaction, p-nitroaniline, produces a strongly yellow-colored solution. The coloration can be followed kinetically by the increase in light absorption at 405 nanometers (nm). The same substrate has been employed in an assay method in which the cleavage product is diazotized by nitrous acid and then coupled with 8-hydroxyquinoline sulfate (U.S. Pat. No. 3,878,048). The substrate has the disadvantage that it is very poorly soluble in the aqueous media employed for assay, and therefore an elevated temperature (60.degree. C) and/or a surfactant must be used to solubilize the substrate. Owing to the low solubility, the assay is performed in the presence of limiting quantities of the substrate.
The solubility of the substrate was increased by the introduction of a sulfonic group in the 3-position of the p-nitroaniline, to provide gamma-glutamyl-3-sulfonic acid-4-nitroanilide (German published application No. 23 33 798), and by the introduction of a carboxyl group in the 3-position, to provide gamma-glutamyl-3-carboxy-4-nitroanilide (German published application No. 22 59 512; see also J. Clin. Chem. Clin. Biochem. 14, 421 (1976). When the assay is followed kinetically, the products released have an absorbance in the range of 350-450nm. In this range the absorbance of hemolyzed, icteric or turbid samples represent a strong interference. This is a definite disadvantage. Furthermore, stability of reagents in solution is limited, as reported in the latter paper.
The solubilities of the several compounds as based upon the literature are as follows:
______________________________________ Substrate Solubility, g./liter ______________________________________ gamma-glutamyl-p-nitroanilide 1-1.25 gamma-glutamyl-3-sulfonic-4- nitroanilide, as NH.sub.4 salt 100-125 gamma-glutamyl-3-carboxylic-4- nitroanilide, as di-NH.sub.4 salt 20-25 ______________________________________
A similar assay method employed gamma-glutamyl aniline as the substrate (Arch. Biochem. and Biophys., 91, 61 (1960)). The aniline cleavage product was diazotized and coupled with N-(1-naphthyl)ethylenediamine in the form of its dihydrochloride addition salt, by a modified Bratton-Marshall procedure (J. Biol. Chem., 128, 537 (1939)). This substrate, however, exhibits poor stability in solution. It also requires heat and/or a surfactant to solubilize the substrate. Owing to the low solubility, the assay is performed in the presence of limiting quantities of the substrate. The upper limit of linearity is low (up to 130 milliunits of enzyme activity per milliliter) and, therefore, frequent dilution of samples with high activity is required.
Another synthetic peptide substrate which has been employed is gamma-glutamyl-alpha-naphthylamide (Clin. Chim. Acta, 14, 619 (1966)). The cleavage product of enzyme reaction is alpha-naphthylamine, which is coupled to the diazonium compound, Fast Blue B salt, to form a strongly colored dye. The development of the color is a relatively slow process, and alpha-naphthylamine has been found to be carcinogenic.